It seems that supercritical extraction could be an interesting method for you.

Please check the below sites and see whether such articles will be helpfull:

1. http://www.clinchem.org/content/32/5/874.short

2. http://www.sciencedirect.com/science/article/pii/S0260877404001372

3. http://www.sciencedirect.com/science/article/pii/S0896844606000866

4. http://www.sciencedirect.com/science/article/pii/S0889157505000712

5. http://www.sciencedirect.com/science/article/pii/S0308814610005492

6. http://agris.fao.org/agris-search/search/display.do?f=1996/KR/KR96012.xml;KR9504044

Exposure to light should be a minimum during the entire extraction process, and amber tinted glassware should be used. I also use an antioxidant like pyrogallol, BHT or Vitamin C in the extraction. Alpha carotene has a very similar UV spectrum, and an isocratic run on a C18 will no doubt have the two eluting on top of each other. As far as the low temp, this may not extract all of the beta carotene. What I have always done is expose the standard to all of the same conditions as the sample, run it through the extraction procedure. A good, though not absolute, way of gauging your extraction is to prepare your samples in triplicate, but spike the third with a known amount of standard prior to extraction and see what your recovery is. Finally, beta-carotene is stubborn to get in solution, and if available THF works great. Hope some of this helps.

I used C30 column and using AC and ACN for extrction. During the extraction and keeping you can add 0,1% of BHT which is good antioxidant for retarding the oxidation in the process. about the knowing the method efficient maybe you can use standard of the compound. I haven't done that but maybe using a certain concentration of the standard in one sample, that you already calculated the concentration of the beta-carotene, can give you some clues.

Estimation of carotenoids has always been challenging.

I answer your questions one by one as per the number you have assigned:

1. I worked in India. We had optimal instrumental facility. I extracted the material using reflux assembly. I knew the method might be objectionable, but my aim was not to estimate absolute quantity in the plant but to put a comparison in between three plants I selected. I optimized the extraction procedure, size of containers, sample amount and volume of solvent needed for maximum extraction of carotenoids. I performed series of pre experiments.

Ideally, it should be done using supercritical extraction (In absence of Heat and solvent exposure), else you may opt maceration in dark for specified hours in most inactive solvent.

2. Beta carotene is not photo stable so utmost care has to be taken with this aspect. You have to add antioxidants in standard solution too. You may use fresh solution of standard and sample every time too.

3. I worked on beta carotene. I got it from local India company, High Media. I am not sure about alpha carotene. Have you checked Sigma- Aldrich etc.?Find the literature where alpha cartoene estimation is given and find the source of standard from those.

UV spectra may not differentiate these two. you need at least IR spectra and match fingerprint region (Superimposing them) with standard IR Spectra. Get IR reference spectra of both alpha and beta carotene.

I had no expertise in the field of carotenoids, I worked accidentally on them. My work was on the constituents responsible for lactogenic properties of the plants and develop the physico chemical standards to distinguish few controversial ayurveda plants.

As a part of my studies I worked on the carotenoids and developed analytical method using HPTLC as it was the only instrument accessible to me (We had only one HPLC in department and I could not take risk of injecting extract in that system on which more than 20 projects were in continuation), so with the knowledge of the limitation, I opted HPTLC.

My suggestion is to refere AOAC journals. Alpha carotene is not a very new compound, you may get lot many manuscripts for the purpose and adopt the method you find more appropriate. Do not start developing method from the scratch itself.

Why alpha- carotene (Q.1 Why carotene, Q.2 Why not beta carotene, more abundant in plants) ?????? Will you be in condition to justify the selection of marker? These are few of the questions, reviewers may ask you. Contact me if you have any further queries.

Best Luck

Some of your questions have straight-forward answers and some of the responses that you have received are misleading. I have worked with carotenoids in foods and biological matrices for 25 years.

1 It is not essential to keep the sample cold during extraction but is a nice precaution. Minimizing exposure to strong UV light and exposure to oxygen is beneficial. Even glassware filters most UV light below 300 nm (eg., place glass cuvettes in spectrophotometer and see the cut-off). Don't work in direct sunlight or in front of windows. We have UV filters on all of our lab lights which only slightly tint the lighting making it easy to work w/o yellow or red lights. Blanket tubes with nitrogen during extraction. Recovery can be estimated (and compensated for) by adding an internal standard during the extraction process. We have used several different compounds over the years (ethyl-B-apo-carotenoate, canthaxanthin, echinenone, etc) - carotenoids that are not in your sample. Antioxidants are beneficial but beware as BHT will polymerize during saponification and show up as a peak at 450nm in your chromatogram. Include samples +/- spikes of beta-carotene to monitor recovery.

2. I have addressed many of the concerns above. Beta-carotene is not as labile as many report. Lycopene is much worse. Carotenoids have differing stability in various solvents. See my J Ag Food Chem pub on relative solubilities. Also note that it may not be stability but solubility. The presence of trace amounts of water in extractions will greatly reduce carotenoid solubility. Ethyl acetate will entrain ~12% water during extraction. Other solvents used have been THF or mixes of THF, acetone, methanol. These will be miscible with the aqueous phase and require further extraction with hexane, pet ether or similar to remove carotenoids from water-miscible solvents.

3 alpha-carotene has been available at various times from Sigma (no longer) Fluka, Atomergic Chemetals,. Currently is should be available from ChromaDex, CaroteNature and DHI.

Since all C18 columns are different, it is not possible to comment on your conditions. a-carotene and B-carotene separate isocratically using most C18 columns. Lutein and zeaxanthin will most likely coleute with the alcohol content in your mobile phase. See my pubs on column and mobile phase combinations. Me tested about 70 columns at NIST.